JP2009100743A - Air-containing fat continuous product - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple and improved process of producing air-containing fat continuous products, and especially to provide the process of obtaining high overrun and uniform and small size bubbles. <P>SOLUTION: Air-containing fat continuous products are provided which contain hydrophobin. The process of producing the products is also provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to fat continuous products such as chocolate and butter. In particular, the present invention relates to an aerated fat continuous food and a method for producing them.

  Fat continuous products such as chocolate, butter, margarine, ghee, oil, shortening, peanut butter and chocolate spread are generally not aerated. However, they can be aerated for various purposes, for example to increase softness and / or stretchability, to change the texture or to change the appearance, such as by whitening or opacification. A well-known example is an aerated chocolate such as Aero ™. Unlike water continuous products such as mousse or ice cream, aeration of fat continuous foods simply by whipping in the presence of a surfactant to high overruns is both hydrophobic for fat and air So it is difficult.

  Chocolate is typically aerated by a method in which a pressurized gas, such as carbon dioxide, is mixed into the melted chocolate. The pressure is then released and the bubbles expand, thereby forming an aerated product. Finally, the aerated chocolate is cooled to solidify the fat and thereby retain the aerated structure. This method has been known for many years, for example from GB 459583 and EP 322952.

  Whipped butter is generally made by whipping air into softened butter at a warm temperature and then cooling it. U.S. Pat. No. 2,937,093 discloses a method for producing whipped margarine. This method involves combining liquid margarine with an inert gas (eg, nitrogen), cooling the mixture, stirring the cooled mixture under pressure to form a flowable mass, and then applying pressure. Including releasing.

  EP 285198 discloses an edible plasticized product, such as margarine or shortening, which contains a fat continuous phase and a dispersed gas phase and exhibits improved spring-up behavior when used in frying. This product is manufactured in a votater line, and the gas is incorporated into the composition near the beginning of the line and while the composition is still largely free of crystallized fat.

US Pat. No. 5,202,147 applies a pressure of about 200 to about 500 psi to a molten mass of peanut butter, rapidly cools it to about 35 ° to about 50 ° F., and inert gas in the molten mass. And then passing the chilled mass through a narrow orifice is disclosed.
British Patent No. 459583 European Patent No. 322952 U.S. Pat. No. 2,937,093 European Patent No. 285198 US Pat. No. 5,202,147 European Patent Application No. 1623631 International Publication No. 01/74864 International Publication No. 96/41882 International Publication No. 01/57076 International Publication No. 00/58342 Balley's Industrial Oil and Fat Products, 8th edition, Shahidi and Fereidoon (ed.), 1-6, 2005, John Wiley & Sons Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Ausubel et al., Short Protocols in Molecular Biology (1999) 4th edition, John Wiley & Sons, Inc. and full edition Current Protocols in Molecular Biology. Wessels, 1997, Adv. Microb. Physio. 38: 1-45. Wosten, 2001, Annu Rev. Microbiol. 55: 625-646 De Vocht et al., 1998, Biophys. J. 74: 2059-68. Wosten et al., 1994, Embo. J. 13: 5848-54. Talbot, 2003, Curr. Biol, 13: R696-R698 MacCabe and Van Alfen, 1999, App. Environ. Microbiol 65; 5431-5435 Wosten, 2001, Annu Rev. Microbiol. 55: 625-646 Collen et al., 2002, Biochim Biophys Acta. 1569: 139-50 Calonje et al., 2002, Can. J. Microbiol. 48: 1030-4 Askolin et al., 2001, Appl Microbiol Biotechnol. 57: 124-30 De Vries et al., 1999, Eur J Biochem. 262: 377-85. Linder et al., 2001, Biomacromolecules Vol. 2, 511-517.

  However, such methods are complex and inconvenient and often result in relatively low overruns and / or large air bubbles. Thus, there remains a need for a simple and improved method of producing aerated fat continuous products, and in particular a method that results in high overruns and uniformly sized small bubbles.

  In our European Patent Application No. 1623631, we have found that the fungal protein previously named hydrophobin has an aqueous foam with excellent stability against disproportionation and coalescence. It has been found to be possible to manufacture. However, only water-continuous aerated food is disclosed. Surfactants / aeration agents used to produce an aerated water continuous product are usually not surface active in non-polar solvents such as fat / oil. The present inventors have now found that a continuous product containing aerated fat can be produced by using hydrophobin. The resulting overrun is high, the bubbles are small and the size is relatively uniform.

  Accordingly, in a first aspect, the present invention provides a continuous aerated fat product comprising hydrophobin.

  Preferably the product is a food product, more preferably the product is selected from chocolate, butter, ghee, margarine, low fat spread, cooking fat and oil, shortening, peanut butter, and chocolate spread.

  Preferably the product comprises at least 0.001% by weight hydrophobin.

  Preferably the product contains at most 1% by weight of hydrophobin.

  Preferably the hydrophobin is in isolated form.

  Preferably the hydrophobin is soluble in water.

  Preferably the hydrophobin is a class II hydrophobin.

  Preferably the product has an overrun of 5-150%, more preferably 10-120%, most preferably 20-100%.

  Preferably at least 50% of the bubbles have a diameter of less than 0.1 mm.

Furthermore, we can use a particularly simple method to provide a continuous aerated fat product by using hydrophobin, which method is small with high overrun and uniform size. Found to bring bubbles. Accordingly, in a second aspect, the present invention provides a method for producing an aerated product according to the first aspect of the present invention, which method comprises the following steps:
a) containing an aqueous composition comprising hydrophobin to form a foam;
b) mixing the foam into the fat continuous composition;
c) optionally cooling the mixed composition.

  In one embodiment, the foam is mixed into the fat continuous composition after being dried, for example by spray drying or freeze drying. The drying method is such that the foam is not destroyed during drying. The fat continuum composition must be soft enough or liquid so that the foam can be incorporated. The fat is then solidified by cooling.

In a third aspect, the present invention provides an alternative method for producing an aerated product according to the first aspect of the present invention, which method comprises the following steps.
a) dispersing a gas in a fat continuous composition containing hydrophobin;
b) optionally cooling the resulting composition.

  The fat continuous composition must be sufficiently soft or liquid so that the gas can be entrained to form a foam. The fat is then solidified by cooling.

  In one embodiment, steps a) and b) are performed simultaneously while the composition is undergoing shear, for example, in a scraped surface heat exchanger or a stirred crystallizer. If the fat continuous composition is sufficiently soft at the time the gas is mixed in, most of the total cooling may be performed prior to step a).

  In another embodiment, the gas is dispersed in the fat continuous composition under pressure and then the pressure is released.

In one variation of the second and third aspects of the present invention, a method of manufacturing an aerated product according to the first aspect of the present invention includes:
a) forming an oil-in-water emulsion;
b) cooling the emulsion while applying shear so that phase inversion of the emulsion occurs;
c) including the emulsion during stage a) and / or stage b).

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Definitions and explanations of various terms and techniques used in fat continuous food systems can be found in Balley's Industrial Oil and Fat Products, 6th edition, Shahidi and Fereidoon (ed.), 1-6, 2005, John Wiley & Sons. Has been. Standard techniques used in molecular and biochemical methods are Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY and Ausubel et al. Short Protocols in Molecular Biology (1999) 4th edition, John Wiley & Sons, Inc., and can be found in the full edition of the book Title Current Protocols in Molecular Biology.

  Unless otherwise specified, all percentages except percentages used for overrun are weight percent.

Hydrophobin Hydrophobin is one of the well-defined proteins (Wessels, 1997, Adv. Microb. Physio. 38: 1-45; Wosten, 2001, Annu Rev. Microbiol. 55). : 625-646), capable of self-assembly at the hydrophobic / hydrophilic interface, and has a conserved sequence:
_{X n -C-X 5~9 -C-} C-X 11~39 -C-X 8~23 -C-X 5~9 -C-C-X 6~18 -C-X m ( SEQ ID NO: 1 )
In the formula, X represents any amino acid, and n and m independently represent one integer. Usually, hydrophobins have up to 125 amino acids. The cysteine residue (c) in the conserved sequence is part of a disulfide bridge. In the context of the present invention, the term hydrophobin has a broader meaning and is also a functionally equivalent protein that exhibits self-assembly characteristics at a hydrophobic-hydrophilic interface and results in a protein film, such as the following sequence: Proteins containing:
_{X n -C-X 1~50 -C-} X 0~5 -C-X 1~100 -C-X 1~100 -C-X 1~50 -C-X 0~5 -C-X 1~ _50- C- _Xm (SEQ ID NO: 2)
Or these include also moieties that exhibit self-assembling characteristics at the hydrophobic-hydrophilic interface, resulting in a protein film. According to the definition of the present invention, self-assembly adsorbs proteins to Teflon and uses secondary structure (generally α-helix) using circular dichroism (De Vocht et al., 1998, Biophys. J. 74: 2059-68).

  Film formation can be achieved by incubating a Teflon sheet in protein solution followed by at least three washes with water or buffer (Wosten et al., 1994, Embo. 13: 5848-54). The protein film can be visualized by any suitable method, such as by labeling with a fluorescent marker or using fluorescent antibodies, as is well established in the art. m and n usually have values ranging from 0 to 2000, but more usually the sum of m and n is less than 100 or 200. The definition of hydrophobin in the context of the present invention includes not only a fusion protein of hydrophobin and one other polypeptide, but also a conjugate of hydrophobin and another molecule such as a polysaccharide.

  Hydrophobins identified to date are generally classified as either class I or class II. Both types have been identified in fungi as secreted proteins that self-assemble at a hydrophobic interface into an amphiphilic film. Class II hydrophobin populations are readily soluble in various solvents, whereas class I hydrophobin populations are generally relatively insoluble. Preferably the hydrophobin is a class II hydrophobin. Preferably the hydrophobin is soluble in water, meaning that it is at least 0.1%, preferably at least 0.5% soluble in water. At least 0.1% soluble means that no hydrophobin precipitates when 0.1 g of hydrophobin in 99.9 mL of water is centrifuged at 30,000 g for 30 minutes at 20 ° C. .

  Hydrophobin-like proteins (eg “chaplins”) are (Actinomycetes) and Streptomyces sp. (International Publication No. 01/74864; Talbot, 2003, Curr. Biol, 13: R696-R698). These bacterial proteins, in contrast to fungal hydrophobins, can only form up to one disulfide bridge because they can only have two cysteine residues. Such a protein is an example of a functional equivalent to hydrophobin having the consensus sequence shown in SEQ ID NOs: 1 and 2, and is within the scope of the present invention.

  Hydrophobins can be obtained by extraction by a suitable method from natural sources such as filamentous fungi. For example, hydrophobins can be obtained by culturing filamentous fungi that secrete hydrophobin into the growth medium or by extraction with 60% ethanol from fungal hyphae. It is particularly preferred to isolate hydrophobins from a host organism that naturally secretes hydrophobins. Preferred hosts are nematodes (eg Trichoderma), basidiomycetes and ascomycetes. Particularly preferred hosts are food grade organisms, such as Chryphonectria parasitica, which secretes one hydrophobin, termed criparin (MacCabe and Van Alfen, 1999, App. Environ. Microbiol 65; Pp. 5431-5435).

  Alternatively, hydrophobins can be obtained through the use of recombinant techniques. For example, host cells can be modified to express normal microbes to express hydrophobins, which can then be isolated and used according to the present invention. Techniques for introducing nucleic acid constructs encoding hydrophobins into host cells are well known in the art. More than 34 genes encoding for hydrophobins have been cloned from more than 16 fungal species (eg, published international publications showing sequences of hydrophobins identified in mushrooms (Agaricus bisporus) 96/41882; and Wosten, 2001, Annu Rev. Microbiol. 55: 625-646). Recombination techniques can also be used to alter the sequence of hydrophobins or to synthesize novel hydrophobins having the desired improved properties.

  Usually, a suitable host cell or host organism is transformed with a nucleic acid construct encoding the desired hydrophobin. Nucleotide sequences encoding polypeptides may be inserted into appropriate expression vectors encoding elements necessary for transcription and translation in such a way that they are expressed under appropriate conditions. (E.g. with appropriate target and expression sequences in the correct orientation and correct reading frame). The methods necessary to construct these expression vectors are well known to those skilled in the art.

  Several expression systems can be used to express the polypeptide coding sequence. These include, but are not limited to, bacteria, fungi (including yeast), insect cell lines, plant cell culture systems and plants all transformed with a suitable expression vector. Preferred hosts are considered food grade-"generally considered safe" (GRAS).

  Suitable fungal species include yeasts such as the genus Saccharomyces, the genus Kluyveromyces, the genus Pichia, the genus Hansenula, the genus Candida, the genus Schizosactomyces (But not limited to), and Filamentous species, such as Aspergillus, Trichoderma, Mucor, Neurospora, Fusarium, etc. (But not limited to).

  The sequence encoding the hydrophobin is preferably at least 80% identical at the amino acid level, more preferably at least 95% or 100% identical to a naturally recognized hydrophobin. However, one skilled in the art can make conservative substitutions or other amino acid changes that do not reduce the biological activity of the hydrophobin. For the purposes of the present invention, these hydrophobins having a high level of identity with naturally occurring hydrophobins are also encompassed within the term “hydrophobin”.

  Hydrophobin can be purified from the culture medium or cell extract by, for example, the procedure described in WO 01/57076, which procedure takes hydrophobin present in the hydrophobin-containing solution on the surface. Adsorbing and then contacting the surface with a surfactant such as Tween 20 to elute the hydrophobin from the surface. Collen et al., 2002, Biochim Biophys Acta. 1569: 139-50; Calonje et al., 2002, Can. J. Microbiol. 48: 1030-4; Askolin et al., 2001, Appl Microbiol Biotechnol. 124-30; and De Vries et al., 1999, Eur J Biochem. 262: 377-85.

  The amount of hydrophobin present in the product generally varies depending on the formulation and the volume of the gas phase. Usually the product contains at least 0.001% by weight of hydrophobin, more preferably at least 0.005 or 0.01% by weight. Typically, the product contains less than 1 wt% hydrophobin, more preferably less than 0.5 wt%, for example about 0.1 wt%. The hydrophobin can be from a single source or from multiple sources, and can be, for example, a mixture of two or more different hydrophobins.

  Hydrophobin is added in a form and in an amount such that it can be used to stabilize the gas phase, i.e. hydrophobin is intentionally added to the product for the purpose of utilizing its foam stabilizing properties. To be introduced. Thus, if a component is present or added that includes fungal contaminants that may contain a hydrophobin polypeptide, this does not constitute adding hydrophobin within the spirit of the invention.

  Usually the hydrophobin is in an isolated form in the product of the invention, usually at least partially purified and added eg at least 10% pure relative to the weight of the solid. By “isolated form” is meant that hydrophobin is not added as part of a naturally occurring organism such as a mushroom that naturally expresses hydrophobin. Rather, hydrophobins are usually either extracted from naturally occurring sources or obtained by expression of recombinants in the host organism.

Fat continuous products Fats are generally triglycerides, ie triesters of glycerin and fatty acids. The term “fat” includes not only fat that is solid, but also oil that is liquid at room temperature.

  The fat continuous product is preferably a food such as chocolate, chocolate analogue, chocolate spread, butter, ghee, margarine / spread, boiled / fried oil, shortening, peanut butter. Fats commonly used in foods are coconut oil, palm oil, palm kernel oil, cocoa butter, milk fat, sunflower oil, safflower oil, olive oil, flaxseed oil, soybean oil, rapeseed oil, walnut oil, corn oil, grape Seed oil, sesame oil, malt oil, cottonseed oil, crushed nut oil, fish oil, almond oil, sesame oil, watermelon seed oil, rice oil, peanut oil, pistachio oil, hazelnut oil, corn oil, and mixtures, fractions or hydrogen Includes foods such as chemicals.

  As used herein, the term “chocolate” includes dark chocolate, white chocolate, and milk chocolate; the term “chocolate analogue” refers to fats other than cocoa butter (eg, cocoa butter equivalents, coconut oil or others Confectionery compositions based on chocolate-like fats made of vegetable oils. Chocolate and chocolate analogs can include cocoa powder, milk solids, sugar or other sweeteners and flavors.

  The terms “margarine” and “spread” refer to many different butter substitutes consisting of water-in-oil emulsions made from vegetable and / or animal fats. In addition to emulsions, margarine / spreads can contain milk proteins, salts, emulsifiers, colorants, flavors and the like. These terms also cover a blend of margarine and butter and a fat continuous low fat spread that usually contains less than 40% fat.

  Shortenings are edible fat products that usually contain close to 100% fat and are prepared from animal and / or vegetable oils. Shortening is used in frying, boiling, baking, and as an ingredient in fillings, icing and other confectionery items.

  In addition to hydrophobins and fats, the aerated foods of the present invention are other ingredients conventionally found in foods such as sugars, salts, proteins, fruits and / or plant materials, emulsifiers, stabilizers, preservatives, colorants. , Flavors and acids can be included.

Aeration and overrun The term “aeration” means having a gas intentionally incorporated in the product, for example by mechanical means. The gas can be any gas, but in the context of food, is preferably a food grade gas such as air, nitrogen, nitrous oxide, or carbon dioxide. The degree of aeration is measured for the “overrun” defined below:

  (Overrun) = {(Weight of non-aerated mixture) − (Weight of aerated product)} × 100 / (Weight of aerated product)

In the formula, the weight relates to a certain volume of the aerated product and the aerated mixture from which the product is made. Overrun is measured at atmospheric pressure.

  Preferably the food product has an overrun of at least 5%, more preferably at least 10%, most preferably at least 20%. Preferably the food product has an overrun of at most 150%, more preferably at most 120%, most preferably at most 100%. In one embodiment, the food is aerated butter, margarine, or spread, where the overrun is preferably 5-50%, more preferably 10-20%, such as about 15%. In another embodiment, the food is cooking oil that is aerated to reduce splashing, in which case the overrun is preferably less than 10%, usually about 5%.

  In one embodiment, the bubbles are small enough so that they are not visible to the naked eye. This has the advantage that it is not aerated as the product can be seen, has the same appearance as a non-aerated product and is preferred by consumers. (Still this aerated product may be slightly lighter or more milky white due to light scattering by small bubbles). For example, chocolate can be aerated and thus has a much lower caloric content per unit volume, but looks similar to unaerated chocolate. Preferably, at least 50% of the bubbles have a diameter of less than 0.1 mm, more preferably less than 0.05 mm (determined from the normalized cumulative frequency as described in Examples 1 and 2 below).

  The invention will now be described in more detail with reference to the following illustrative and non-limiting examples and figures.

Example 1
Chocolate aerated by addition of foam Chocolate having the formulation shown in Table 1 was heated to 45 ° C.

  Hydrophobin HFB II was obtained from VTT Biotechnology, Finland. It was purified from Trichoderma reesei essentially as described in WO 00/58342 and Linder et al., 2001, Biomacromolecules Volume 2, pages 511-517. 20 ml of a 0.05 wt% aqueous solution of HFB II was aerated to 50 ml volume using an Aerolatte battery powered handheld whisk (Aerolate Ltd, Radlett Hertfordshire, UK). The rotor of the whisk was a horizontal circular wire coil having an outer diameter of 22 mm and rotated around a vertical axis passing through the center thereof at a rotational speed of about 12000 rpm. The free water was removed by pipette and discarded to drain the foam and minimize the amount of water added to the chocolate after 10 minutes (even the addition of a small amount of water affects the quality of the chocolate texture. It is known). The foam was then folded into molten chocolate using a metal pallet knife to form 100 ml of aerated chocolate.

  For comparison, an aerated chocolate was prepared using a foam stabilized with a conventional food aeration agent Hygel (Kerry Foods, Ireland) instead of hydrophobin. A high gel sample was prepared in the same manner except that the foam was used immediately without draining. 12.4 ml of 0.8% by weight high gel solution was foamed and mixed into 50 ml of molten chocolate.

  The overrun was determined by weighing a certain volume of unaerated and aerated chocolate. The aerated chocolate was poured into a mold and cured at −10 ° C. for 2 hours. The texture and appearance of the chocolate was evaluated. Both products had a soft texture and there were no common weaknesses associated with chocolate. The high gel sample had a crumbly texture, while the hydrophobin sample had a truffle-like texture, again fragile but with a smoother appearance. The high gel sample had a relatively low overrun and some air bubbles could be seen with the naked eye. The hydrophobin sample had a higher overrun and the bubbles were small and not visible. The results are summarized in Table 2.

The microstructure of each product was visualized using a cryogenic scanning electron microscope. Each sample was cooled to −80 ° C. on dry ice, a section about 5 mm × 5 mm × 10 mm in size was cut out and Tissuesue Tek OCT ™ compound (PVA 11%, Carbowax 5) % And non-reactive components 85%) were attached to the sample holder. The sample containing the holder was placed in a liquid nitrogen slush and transferred to a cryogenic chamber (Oxford Instruments CT1500HF). The chamber was kept under a vacuum of about ^10-4 bar. The sample was warmed to −90 ° C. and held for 60-90 seconds, then cooled to −110 ° C. and then coated with gold using an argon plasma at a pressure of 10 ⁻¹ mbar and a current of 6 mA for 45 seconds. Finally, the sample was transferred to a scanning electron microscope (JSM5600) fitted with an Oxford Instruments cold stage maintained at a temperature of -160 ° C. Samples were examined and representative areas were captured with digital image acquisition software.

  FIG. 1 shows an SEM image. The hydrophobin sample contains a lot of small air bubbles (less than 50 μm in diameter), while the high gel sample has fewer and larger air bubbles. Example 1 shows that the use of hydrophobin results in higher overruns and smaller bubbles than an equivalent chocolate made with conventional food aerating agents. This air structure is soft and gives the chocolate a truffle-like texture.

(Example 2)
Chocolate aerated with carbon dioxide 45 ° C. chocolate (having the formulation presented in Table 1 above) was poured into a 75 ml aerosol can. 1 g of 100 mg / ml HFB II aqueous solution was added to the chocolate and mixed with shaking. As a comparison, an aerosol can containing chocolate having 1 g of pure water and chocolate alone was prepared. The can was sealed, shaken, pressurized to 4 bar with carbon dioxide and shaken again. The chocolate containing the HFB solution and water in these cans is observed to be thicker than pure chocolate when shaken, and the addition of water may have a thickening effect on the chocolate. As it was known, it was as expected. These cans were stored overnight at 45 ° C. and repressurized to 4 bar with carbon dioxide. These contents are then valved (Precision Valve, Peterborough, UK; with two 3.2 × 4.6 mm orifices in a standard 1 inch cup, and with four slots and one tail piece orifice. Dispensed into a plastic pot through a 4.8 mm stem with housing). Relieving the pressure by opening the valve causes the bubbles to expand and form an aerated chocolate. The filled pot was immediately placed in a blast freezer and stored at −25 ° C. overnight. After storage, the chocolate sample was crushed and the bubble structure was observed and photographed.

The density of chocolate was measured as follows. Two liters of water (4 ° C.) was placed in a beaker on the balance. The scale was then measured. A piece of chocolate (about 30 g) was placed next to the beaker on the scale and weighed (m ₁ ). The piece was then held under water using tweezers, taking care not to touch the side or bottom. The balance reading was recorded (m ₂ ). According to Archimedes' principle, the difference in reading before and after immersion (m ₁ -m ₂ ) is equal to the water density multiplied by the water volume. The volume of water displaced is that of chocolate pieces. The density of chocolate is its mass (m ₁ ) divided by its volume. The overrun is calculated as before (using the density in the equation for calculating the above overrun is equivalent to using a constant volume). The measurement was repeated twice and the average was taken. The results are shown in Table 3.

  As expected, in each case, the gas successfully aerated the chocolate to a high overrun (about 60%). However, although the overrun was similar, HFB II had a significant effect on the bubble size and distribution as shown in FIG. Standard chocolate had a wide distribution of bubble sizes and the bubbles were creamed near the surface. In chocolate with water, the bubbles were somewhat small and there was no creaming, probably due to the increased viscosity of the chocolate. In chocolate containing HFB II, there was a much more uniform bubble size distribution and some very small bubbles were observed (although they were larger than in the hydrophobin sample of Example 1). .

  The bubble size distribution of the aerated chocolate sample containing water and HFB II solution was determined from FIGS. 2B and 2C, respectively, using the following method. First, a trained operator (who is familiar with the aerobic microstructure) uses a graphical user interface on a digital image (ie, a two-dimensional representation of a three-dimensional microstructure) to identify bubbles and Trace the outline. The bubble size was calculated as follows from each outline defined by the operator. The maximum bubble area (A) was determined and multiplied by a scaling factor defined by the magnification of the image. The bubble diameter is defined as the equivalent circle diameter d:

  d = 2√ (A / π)

  This is an accurate definition of the diameter of a perfectly spherical two-dimensional cross section. This is a good means of measuring size since most of the bubbles were nearly spherical. The size distribution was obtained by constructing a histogram consisting of bins of width W μm. B (j) is the number of bubbles per unit area in the jth bin (ie in the diameter range j × W to (j + 1) × W). B (j) is obtained by adding all the individual contributions of bubbles having a diameter in the range of J × W to (j + 1) × W. The bubble size distribution is conveniently described as a fraction of the total number of bubbles measured, with respect to the normalized cumulative frequency, i.e. the sum of bubbles having a diameter up to a given size. . The size determination and distribution configuration is conveniently performed automatically on a computer, for example by using the MATLAB® 2006a (MathWorks, Inc) software.

  FIG. 3 shows the normalized cumulative frequency obtained. These demonstrate that the bubbles produced when chocolate is aerated with hydrophobin are smaller than when no hydrophobin is used.

(Example 3)
Aerated butter Butter was made from double cream (40% fat, Dairy Crest Ingredients, UK) by shearing at 15-20 ° C. This causes the fat globules in the cream to stick together and agglomerate, eventually resulting in a phase inversion to fat continuous butter. The mixture is then strained to remove the buttermilk. A soft and pliable butter was obtained that was sufficient to allow the foam to be folded into an aerated product.

  The foam was made by aerating 12 ml of a 9.76 mg / ml HFB II solution to a volume of 80 ml using an Aerolatte instrument. Half of this foam (40 ml) was blended with 72 g of butter. The aqueous high gel foam was made by aerating 12.4 ml of an 8 mg / ml high gel solution to a volume of 60 ml using an Aerolatte instrument. This foam was combined with 76 g of butter. The obtained samples were put in a pot and cured at 5 ° C. for 1 week, and their overrun was measured as in Example 1. The hardness of the aerated butter was also measured using a Brookfield LFRA Texture Analyzer with a cone of 45 ° and a bottom diameter of 30 mm. The cone was pushed into the product at a depth of 2 mm / sec to a depth of 10 mm and the peak load was recorded. Unaerated butter samples were also measured for comparison. Results are presented in Table 4.

  Butter prepared using high gel foam had zero overrun (ie, air was completely lost) and a hardness similar to that of non-aerated butter. The HFB II foam was stable enough to mix into the butter, but some of the overrun was lost. The resulting product was much softer than unaerated butter. The introduction of air means that the butter caloric content (per unit volume) has been reduced by about one third. The air bubbles were too small to be seen with the naked eye, but the aerated butter was whiter than the unaerated butter, indicating the presence of numerous small air bubbles. A photograph of the aerated butter containing non-aerated butter and hydrophobin is shown in FIG.

Example 4
Aerated spread A fat continuous spread (margarine) was prepared using the formulation presented in Table 5.

  The emulsion was prepared as follows. Hard stock fat blend (40% hardened palm oil, 60% hardened palm kernel oil), lecithin and saturated monoglyceride were dissolved in sunflower oil. Whey powder, salt and potassium sorbate were dispersed in hot water and the pH was adjusted to 4.7 using citric acid. The aqueous phase was then added to the fat phase and mixed with a high speed Silverson mixer for 10 minutes to ensure good emulsification. The emulsion was transferred to a jacketed premix tank and stirred. Spreads were prepared from this emulsion using two different methods.

  A portion of the emulsion was processed using a small votor line consisting of the following equipment: HSC (high speed pin mixer), A (surface scraped heat exchanger-SSHE), C (pin mixer), A (SSHE). The first C unit was not cooled, but all subsequent units were cooled to 5 ° C. All devices were operated at a throughput of 50 g / min at 1000 rpm. The product outlet temperature was 15.7 ° C. A sample of unaerobed spread was collected and removed. Another set of samples was obtained at a final HFB II concentration of 0.05% by manually mixing a foamed aqueous solution of HFB II into the spread to 15%, 25% and 50% overrun. All samples were stored cold.

  The second part of the emulsion was processed through a small surface scraping heat exchanger (SSHE) that allowed air to be incorporated during the mixing and cooling steps. This small SSHE consisted of a jacketed cylindrical horizontal barrel (running capacity 145 ml) cooled with silicone oil coolant. The barrel contained a stirrer consisting of two stainless steel scraping blades attached to a shaft rotating around the barrel axis to scrape the cold inner surface of the barrel. The blade was hinged to move freely around the mandrel evenly spaced. The refrigerator contained one inlet / outlet valve, an exhaust valve and a temperature measuring end. The process conditions were as follows: emulsion flow rate 30 ml / min, stirrer speed 910 rpm, jacket temperature 0 ° C., outlet temperature 6.2 ° C. First, a sample not containing air was taken out: air was then introduced at a flow rate of 20 ml / min to take out the aerated sample (overrun 30%). Finally, an aqueous solution of HFB II was added to the emulsion to give a final concentration in the product of 0.05% and a further aerated sample (overrun 33%) was removed. The hardness of the spread was measured as described in Example 3, but using a 6 mm cylinder as a probe instead of a cone. Results are presented in Table 6.

  Table 6 shows that, as expected, inclusion of air softens the spread. This is because the spread made in the small SSHE was harder than the botator sample and the former had finer water droplet distribution. Comparison of the aerated small SSHE sample shows that the sample containing HFB II was softer than the one containing no HFB II. This is due in part to the slightly higher overrun of the HFB II sample, but in part due to their air structure differences. Confocal microscopy showed that the air bubbles in the sample containing HFB II were more uniform. Small SSHE samples were spread on oil-resistant paper and evaluated visually. The sample containing HFB II resulted in a smoother surface after spreading than the aerated sample containing no HFB II.

(Example 5)
Aerated liquid margarine Two aqueous solutions were prepared: the first contained 0.25% by weight HFB II and the second contained 0.25% by weight Higel. Foams were made by aeration of 100 ml of each solution to the maximum air phase volume. This was done by first using a hand-held Bamix blender and then using a hand-held Aero-latte instrument to refine the air bubbles. The foam was then drained so that as little water as possible was brought into the final product. The HFB II foam was drained for 30 minutes, but the high gel solution required only a few minutes.

  An aerated liquid margarine was then prepared by gently mixing 34 ml of each foam into 66 ml of Blue Band liquid margarine (Unilever, UK) to make a product with a 50% target overrun.

  Upon incorporation into liquid margarine, the high gel foam lost some air phase volume resulting in a reduced final volume (˜90 ml) and small air bubbles were visible. In contrast, the HFB II foam was well incorporated into liquid margarine and did not lose any air. After 5 days storage, the HFB II sample did not lose any volume.

(Example 6)
Aerated and tempered chocolate Two aqueous foams were prepared as described in Example 5 using HFB II and Higel. Milk chocolate (Cadbury's Dairy milk) was placed in a beaker and gently warmed on a larger warm water (55 ° C.) container. The chocolate was gently agitated to ensure uniform melting. The final temperature was about 30 ° C. (ie, warm enough to fluidize the chocolate without melting all of the fat). 75 ml of the drained foam was gently folded into a 100 g portion of molten chocolate until the mixture was uniform. The sample was filled into a 30 ml plastic pot and allowed to cool to room temperature to maintain tempered condition.

  Upon incorporation into chocolate, the high gel foam collapsed, losing much of the air phase volume, and the final overrun was reduced to only 3.6%. In contrast, the HFB II foam was evenly mixed in the chocolate and the chocolate had a final overrun of 16%.

  In summary, Examples 1-6 demonstrate that continuous aerated fat products such as chocolate, butter, and spread can be successfully manufactured by using hydrophobins. The resulting product has improved properties (smaller and / or more uniform air bubbles and / or higher overrun) compared to an aerated product that does not use hydrophobin.

  The various features and embodiments of the invention mentioned in the individual sections above apply mutatis mutandis to the other sections. Thus, features specified in one section can be combined with features specified in other sections as appropriate. All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and product of the invention will be apparent to those skilled in the art without departing from the scope of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

It is a scanning electron micrograph of the microstructure of (a) chocolate aerated with a foam of HFB II and (b) chocolate aerated with a high gel foam. It is a photograph of the structure of chocolate aerated with carbon dioxide: (a) chocolate; (b) chocolate using water; (c) chocolate using hydrophobin solution. It is a graph which shows the normalized accumulation frequency as a function of a bubble diameter about the aeration chocolate shown to FIG.2 (b) and (c). 2 is a photograph of (a) unaerated butter and (b) an aerated butter containing hydrophobin.

Claims (15)

  Continuous product of aerated fat containing hydrophobin.   The product of claim 1 which is a food product.   The product of claim 2, wherein the food is selected from chocolate, butter, ghee, margarine, low fat spread, cooking fat and oil, shortening, peanut butter, and chocolate spread.   4. A product as claimed in any one of the preceding claims comprising at least 0.001% by weight of hydrophobin.   5. Product according to any one of claims 1 to 4, comprising at most 1% by weight of hydrophobin.   The product according to any one of claims 1 to 5, wherein the hydrophobin is in an isolated form.   The product according to any one of claims 1 to 6, wherein the hydrophobin is soluble in water.   The product according to any one of claims 1 to 7, wherein the hydrophobin is a class II hydrophobin.   9. A product according to any one of the preceding claims having an overrun of 5 to 150%.   10. Product according to any one of the preceding claims, wherein at least 50% of the bubbles have a diameter of less than 0.1 mm. a) containing an aqueous composition comprising hydrophobin to form a foam;
b) mixing the foam into a fat continuous composition;
c) A method for producing a continuous aerated fat product comprising hydrophobin, comprising the step of optionally cooling the mixed composition.   12. The method of claim 11, wherein the foam is dried and then mixed into the fat continuous composition. a) dispersing a gas in a continuous fat composition containing hydrophobin;
b) A process for producing a continuous aerated fat product comprising hydrophobin, comprising optionally cooling the resulting composition.   14. The method of claim 13, wherein steps a) and b) are performed simultaneously while the composition is undergoing shear.   15. A method according to claim 14, wherein in step a) a gas is dispersed under pressure in the fat continuous composition and then the pressure is released.